simple,automated,highresolutionmassspectrometry ...viral glycoprotein-receptor interaction itself...

Simple, Automated, High Resolution Mass SpectrometryMethod to Determine the Disulfide Bond and GlycosylationPatterns of a Complex ProteinSUBGROUP A AVIAN SARCOMA AND LEUKOSIS VIRUS ENVELOPE GLYCOPROTEIN*

Received for publication, February 10, 2011, and in revised form, March 21, 2011 Published, JBC Papers in Press, March 23, 2011, DOI 10.1074/jbc.M111.229377

Gennett M. Pike‡1, Benjamin J. Madden§1, Deborah C. Melder‡, M. Cristine Charlesworth§, and Mark J. Federspiel‡2

From the ‡Department of Molecular Medicine, §Mayo Proteomics Research Center, the Mayo Clinic, Rochester, Minnesota 55905

Enveloped virusesmust fuse the viral and cellularmembranesto enter the cell. Understanding how viral fusion proteinsmedi-ate entry will provide valuable information for antiviral inter-vention to combat associated disease. The avian sarcoma andleukosis virus envelope glycoproteins, trimers composed of sur-face (SU) and transmembrane heterodimers, break the fusionprocess into several steps. First, interactions between SU and acell surface receptor at neutral pH trigger an initial conforma-tional change in the viral glycoprotein trimer followed by expo-sure to low pH enabling additional conformational changes tocomplete the fusion of the viral and cellular membranes. Here,we describe the structural characterization of the extracellularregion of the subgroup A avian sarcoma and leukosis virusesenvelope glycoproteins, SUATM129 produced in chicken DF-1cells. We developed a simple, automated method for acquiringhigh resolution mass spectrometry data using electron capturedissociation conditions that preferentially cleave the disulfidebondmore readily than the peptide backbone amide bonds thatenabled the identification of disulfide-linked peptides. Seven ofnine disulfide bonds were definitively assigned; the remainingtwobondswere assigned to an adjacent pair of cysteine residues.The first cysteine of surface and the last cysteine of the trans-membrane form a disulfide bond linking the heterodimer. Thesurface glycoprotein contains a free cysteine at residue 38 pre-viously reported to be critical for virus entry. Eleven of 13 pos-sible SUATM129 N-linked glycosylation sites were modifiedwith carbohydrate. This study demonstrates the utility of thissimple yet powerful method for assigning disulfide bonds in acomplex glycoprotein.

To enter cells and begin replication, enveloped viruses mustfuse the membrane coating the viral particle with a cellularmembrane to deliver a subviral particle inside the cell (forreview, see Refs. 1, 2). The fusion of twomembranes is thermo-dynamically favored but comes with a very high kinetic barri-er(s). Enveloped viruses have one or more glycoproteins tomediate the fusion process using the energy liberated uponconformational changes of the viral glycoprotein(s) to clear thekinetic barrier(s). Viral entry begins when the viral glycopro-

teins bind an appropriate cell surface protein initiating oneof three possiblemechanistic fates. The viral glycoprotein-recep-tor interaction can serve to enable trafficking of the virion intothe endocytic pathway where the low pH environment triggersa conformation change in the viral glycoproteins initiatingfusion of the viral and endosome membranes (e.g. influenzavirus). With other viruses, for example most retroviruses, theviral glycoprotein-receptor interaction itself triggers a confor-mational change in the viral glycoproteins at neutral pH at thecell surface sufficient for the ultimate fusion of the viral andcellular membranes. In a third mechanism, the interactionbetween the viral glycoprotein and receptor at neutral pH at thecell surface triggers an initial conformational change in the viralglycoproteins. The triggered glycoproteins then require expo-sure to low pH to complete the conformational changes forfusion of the viral and cellular membranes (e.g. avian sarcomaand leukosis viruses (ASLVs)).3 Understanding how viral fusionproteins mediate entry will provide valuable information forantiviral intervention to combat associated disease. We areusing the homologous group of retroviruses, subgroups A to EASLV, to study enveloped virus entry because these viruseshave evolved from a common ancestor to use different cellularproteins as receptors (3, 4) and have maintained the fusionprocess to efficiently enter cells (5–7).All retroviral glycoproteins are synthesized as polyprotein

precursors consisting of the surface glycoprotein (SU) that con-tains the domains that bind the cellular receptor and the trans-membrane glycoprotein (TM) that anchors the protein to themembrane and contains the domains responsible for the fusionprocess (for review, see Ref. 8). After synthesis, the precursorpolyproteins are glycosylated and form trimers through nonco-valent interactions between extracellular regions of theTMgly-coprotein. The viral glycoprotein precursors form a mature,metastable complex capable of mediating virus entry into thecell only after the SU and TM domains are cleaved by a cellularprotease to yield a trimer of SU/TM heterodimers (9). Theinteraction ofmultiple, noncontiguous regions of SU and a spe-cific receptor protein are required to initiate the entry processby triggering a conformational change in the SU glycoproteins,

* This work was supported, in whole or in part, by National Institutes of HealthGrant AI48682. This work was also supported by the Mayo Clinic.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Mayo Clinic, 200 First

Street, SW, Rochester, MN 55905. E-mail: [email protected].

3 The abbreviations used are: ASLV, avian sarcoma and leukosis virus; CID,collision-induced dissociation; ECD, electron capture dissociation; ESI,electrospray ionization; FT, Fourier transform; FTICR, Fourier transformion cyclotron resonance; MS/MS, tandem mass spectrometry; PNGase F,N-glycosidase F; SU, surface; TCEP, tris (2-carboxyethyl) phosphine; TEV,tobacco etch virus; TM, transmembrane.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 20, pp. 17954 –17967, May 20, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

17954 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 20 • MAY 20, 2011

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allowing the kinetically trapped TM glycoprotein structure toextend and form a lower energy structure that projects thefusion peptide toward the target membrane. Two domains inTM are critical for the extension and the subsequent refoldingof TM: the N-terminal and C-terminal heptad repeats. Thelowest energy form of the TM trimer, the six-helix bundle,forms when the C-terminal heptad repeats fold back intogrooves created by the N-terminal heptad repeats, bringing theviral and cellularmembranes into close proximity. Fusion of themembranes goes through an initial outer lipid leaflet mixing,then hemifusion, initial pore formation, pore widening, and thecompletion of fusion where the retroviral glycoprotein 6HBmay undergo additional structural rearrangements (10–12).The cooperation of several glycoprotein-receptor interactionsis likely required to form a local environment capable of fusion.The subgroupA to EASLVenvelope glycoproteins are highly

related: differences in the hypervariable domains of SU (hr1,hr2, and vr3) define receptor usage and suggest that theseviruses have evolved from a common ancestor (13). Tva, thereceptor for subgroup A ASLV, is related to the ligand-bindingrepeat of the low density lipoprotein receptor family (14, 15).Tvb, the receptors for the subgroup B, D, and E ASLVs, is amember of the tumor necrosis factor receptor family (16–18). Tvc, the receptors for subgroup C ASLVs, is related toyet a third disparate family of proteins, the immunoglobulinIg proteins (19). The normal physiological functions of Tva,Tvb, and Tvc are currently unknown. Engineered secretedforms of these ASLV receptors, the extracellular domain ofthe receptor alone or fused to a IgG domain, retain biologicalactivity sufficient to bind the viral glycoproteins with highaffinity and trigger conformational changes in the viral gly-coproteins similar to changes expected to occur during ini-tiation of the infection process (20–22). Engineered secretedforms of the SU glycoproteins either alone or fused to an IgGdomain can bind the soluble forms of the receptors in a sub-group-specific manner (23). The secreted forms of the ASLVSU or its receptor act as competitive inhibitors, having asignificant antiviral effect on the specific ASLV subgroupinfection of cells (22, 24).We are characterizing the structural and functional organi-

zation of the ASLV glycoproteins to understand better how theASLV fusion proteins mediate the entry process (25). Becausethe number and location of the cysteine residues, 14 Cys in SU,5 inTM, and 13 of 14 possibleN-linked glycosylation sites, 12 inSU, 2 in TM, are conserved in the subgroup A to E ASLV gly-coproteins, it is likely these glycoproteins share a commonstructure and glycosylation pattern. Unusual for retroviruses,the ASLV TM glycoprotein has an internal fusion peptide 21residues downstreamof theN terminuswith 2 cysteine residuesflanking the fusion peptide. The organization of the functionaldomains of the ASLV TM fusion protein is remarkably similarto the Ebola virus fusion protein, GP2, although the ASLV SUand Ebola virus GP1 glycoproteins are not (26, 27). A disulfidebond reported to be stable throughout the fusion process cova-lently connects the ASLV SU and TM glycoproteins. If weassume that the disulfide bond links 1 cysteine from SU and 1cysteine from TM, then there is at least 1 cysteine in SU that isan unbound, free cysteine. Recently, Smith and Cunningham

reported that the formation of a reactive cysteine thiolate atresidueCys38 in the subgroupASUglycoproteinwas absolutelyrequired for productive ASLV infection but was not necessaryfor the glycoprotein binding of the Tva receptor or Tva-trig-gered conformational changes (28). Also, therewas no evidencethat the cysteine thiolatemediated isomerization of the SU-TMdisulfide bond.In this study we physically mapped the secondary structure

of an engineered secreted form of the subgroup A ASLV enve-lope glycoprotein (Fig. 1A). Einfeld and Hunter describe asecreted form of the subgroup A envelope glycoprotein trun-cated at TM residue 129 (SUATM129) (Fig. 1B), deleting thetransmembrane and cytoplasmic domains, which was stillcapable of forming oligomers (likely trimers) detected bysucrose gradient (29). The SUATM129 glycoprotein wasexpressed in chicken DF-1 cells (30, 31), a natural cell substratefor ASLV infection and replication and highly purified from cellculture supernatants for this study. We used this biologicallyactive, complex glycoprotein to develop a simple, automatedmethod for acquiring high resolution mass spectrometry datathat enabled the identification of disulfide-linked peptides, theassignment of most of the disulfide bonds, and the identifica-tion of the sites of N-linked glycosylation from a single HPLCrun.

EXPERIMENTAL PROCEDURES

Materials—All of the proteases used, trypsin, chymotrypsin,and Asp-N- endoproteinase, were sequencing grade and pur-chased from Roche Diagnostics. The PNGase F was purchasedfrom New England Biolabs, and the Zwittergent 3-16 fromEMDChemicals (Gibbstown,NJ). The solventswere purchasedfromHoneywell Burdick and Jackson (Morristown,NJ), and thetrifluoroacetic acid (TFA) and formic acidwere purchased fromFluka. The tris (2-carboxyethyl) phosphine (TCEP) and N-eth-ylmorpholine were purchased from Sigma-Aldrich.Cloning and Expression of the SUATM129 Glycoprotein—To

aid in the purification of the SUATM129 glycoprotein, a 10-his-tidine residue tag preceded by the TEV protease cleavagesequence was added in-frame after amino acid 469 of the SR-Aenveloped protein (Fig. 1). An adapter plasmid encoding theSUA-rIgG immunoadhesin, PUCCLA112N-SUA-rIgG, wasdescribed previously (23). The IgG regionwas replacedwith theSalI to MfeI fragment of the SR-A env gene yielding a codingregion that begins with a consensus Kozac start site, NcoI site,and encodes the complete leader region and the SR-A envelopeto residue 469.Oligonucleotideswere then used to add theTEVprotease and His10 sequences by cloning as an MfeI to PstIfragment into PUCCLA112-SUATM129. The complete andtagged SUATM129 expression cassette was isolated as a ClaIfragment and cloned into the TFANEO expression plasmidunder the transcriptional control of SR-A LTRs (32). TFANEOalso encodes a neo expression cassette for selection.DF-1 cells (30, 31)were grown inDulbecco’smodified Eagle’s

medium (Invitrogen) supplemented with 10% fetal bovineserum (Invitrogen), 100 units of penicillin/ml, and 100 �g ofstreptomycin/ml (Quality Biological, Gaithersburg, MD) at39 °C and 5% CO2. DF-1 cells transfected with the TFANEO-SUATM129 plasmidwere grown in 500�g ofG418/ml to select

ASLV Env Disulfide Bond Pattern

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for neomycin-resistant cells. Cloneswere isolated using cloningcylinders (Bellco Glass Inc., Vineland, NJ), expanded, andmaintained with standard medium supplemented with 250�g/ml G418.

Purification of the SUATM129 Glycoprotein—When theSUATM129-expressing cells were confluent, the medium waschanged to a serum-free medium, VP-SFM (Invitrogen) sup-plemented with 4 mM L-glutamine (Invitrogen), harvested 24 h




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later, and cleared of debris using a 3 �M capsule filter (12116;PALL). The cleared supernatantwasmixedwithTALONMetalAffinity Resin (Clontech), prewashed, and equilibrated withwash buffer (50 mM sodium phosphate, pH 7.0, 300 mM NaCl)for 90 min at room temperature to bind the SUATM129 pro-tein His10 tag, and then loaded into a chromatography columnby gravity flow. The resinwaswashedwithwash buffer (500 bedvolumes) and then washed with 2.5 mM imidazole followed by10 mM imidazole in wash buffer (100 bed volumes each). Theresin was then eluted first with 5 bed volumes of 75 mM imid-azole followed by 5 bed volumes of 250 mM imidazole in washbuffer, and the fractions were combined, concentrated and thebuffer exchanged with 100 mM sodium phosphate, pH 7.0,using JumboSep concentrators with 10K filters (PALL). Topurify the SUATM129 protein further from contaminatingproteins, ammonium sulfate was added to the preparation,resulting in a loading buffer of 1 M ammonium sulfate, 100 mM

sodium phosphate, pH 7.0, and loaded onto an octyl-Sepharosecolumn. The column was washed with a gradient exchangingthe load buffer to 100 mM sodium phosphate, pH 7.0, buffer.The SUATM129 protein was eluted first with water, recovering�60–70% of the protein, followed by elution with 20% ethanolthat recovered some additional SUATM129 protein. Fractionscontaining SUATM129 protein were combined and concen-trated, and the buffer was exchanged with 10 mM HEPES, pH7.4, 100 mM NaCl, 5% glycerol for storage.PAGE and Western Transfer Analysis—To analyze reduced

proteins, the samples were adjusted to 1� Laemmli loadingbuffer (2% SDS, 10% glycerol, 50mMTris-Cl, pH 6.8, 5%�-mer-captoethanol, 0.1% bromphenol blue) and boiled for 5 min. Toanalyze nonreduced proteins, the samples were adjusted to 1�Laemmli loading buffer without the �-mercaptoethanol (2%SDS, 10% glycerol, 50 mM Tris-Cl, pH 6.8, 0.1% bromphenolblue) and boiled for 5min. To analyze native proteins, the sam-ples were adjusted to 1� Laemmli loading buffer without theSDS and �-mercaptoethanol (10% glycerol, 50 mM Tris-Cl, pH6.8, 0.1% bromphenol blue) and loaded directly. Proteins wereseparated on Criterion 4–15% Tris-HCl gradient polyacryl-amide gels (Bio-Rad) and either silver stained directly (33), orthe proteins were transferred onto nitrocellulose membranes.TheWestern transfer filterswere blocked in phosphate-buff-

ered saline (PBS) with 10% nonfat dry milk for 1 h at 25 °C. Thefilters were then rinsed briefly in rinse buffer (100 mMNaCl, 10mM Tris-Cl, pH 8, 1 mM EDTA, 0.1% Tween 20) and incubatedwith either an unconjugated anti-His antibody (1:3000 dilution)(27471001; GE Healthcare) or anti-ASLV SU(A) monoclonalantibody (34) (purified from the mc8C5 hybridoma, a kind gift

from Christina Ochsenbauer-Jambor and Eric Hunter, Univer-sity of Alabama at Birmingham, AL) (1:1000 dilution) in rinsebuffer containing 1% nonfat drymilk for 1 h at 25 °C. The filterswere washed extensively with rinse buffer and then incubatedwith 50 ng/ml peroxidase-labeled goat anti-mouse IgG (H�L)(Kirkegaard and Perry, Gaithersburg, MD) in rinse buffer with1%nonfat drymilk for 1 h at 25 °C.After extensivewashingwithrinse buffer, immunodetection of the protein-antibody-perox-idase complexes was performed withWestern blotting Chemi-luminescence Reagent (PerkinElmer Life Sciences). The immu-noblots were then exposed to Kodak X-Omat film.ALV Alkaline Phosphatase Assay—For alkaline phosphatase

assays, DF-1 cell cultures (�30% confluent) were incubatedwith 10-fold serial dilutions of the appropriate RCASBP/alka-line phosphatase virus stocks for 36–48 h. The assay for alka-line phosphatase activity was described previously (22).Protease Digestions and HPLC—Trypsin digestions were

done as follows. 100 �g of SUATM129 was initially digestedwith 2.5 �g of trypsin in 100 mM Tris, pH 8.4, 0.004% Zwitter-gent 3-16 buffer at 55 °C for 2 h. An additional 2.5 �g of trypsinin 100 mM Tris, pH 8.4, 0.004% Zwittergent 3-16 buffer wasthen added to the reaction and incubated at 37 °C overnight.Chymotrypsin digestions were done as follows. 100 �g of puri-fied SUATMwas digestedwith 5�g of chymotrypsin in 100mM

Tris, pH 8.5, 0.004% Zwittergent 3-16 buffer at room tempera-ture overnight. To remove allN-linked glycosylation, PNGase F(500 units/�l) (3000 units) was added to the postdigest reac-tions and incubated at 37 °C for 3 h. Each digest mixture wassplit into two aliquots, with the disulfide bonds reduced in onealiquot by adding TCEP (25 mM final) and incubating at 55 °Cfor 2 h.The nonreduced or reduced peptidemixtures were each sep-

arated with a Zorbax 300 SB-C18, 5-�m, 150� 0.5-mm reversephase column using an Agilent 1100 Series Capillary HPLCsystemat a flow rate of 15�l/min and collecting 1-min fractionsusing gradients described in Table 1. Detection was by UVabsorbance at 214 nm.All fractionswere then dried downusinga SpeedVac spinning concentrator and stored at �80 °C. Frac-tions were reconstituted with 40% acetonitrile, 5% isopropylalcohol, and 0.2% formic acid prior to analysis by direct chip-based infusion Fourier Transform ion cyclotron resonancemass spectrometry using an LTQ-FTICR-MS with electroncapture dissociation (ECD) and an Advion Nanomate 100.EndoproteinaseAsp-Nwas utilized for the fractions found to

contain adjacent cysteines. The digests were performed ondried HPLC fractions that were resolubilized in 100 mM Tris,pH 8.4, with 4 ng ofAsp-N and digested at 37 °C overnight prior

FIGURE 1. Amino acid sequence of the mature subgroup A ASLV envelope glycoprotein and analysis of the purified SUATM129 glycoprotein.A, functional regions of SUA, hr1, hr2, and vr3 primarily responsible for interacting with the Tva receptor, TM, the internal fusion peptide domain, the N- and C-�helices and the transmembrane domain, are indicated. The 19 cysteine residues are numbered and highlighted in red. The 13 possible N-linked glycosylationsites are numbered and highlighted in blue. SUA and TM are cleaved after amino acid 340 (blue arrow). B, schematic representations of the mature wild-typeASLV(A) and SUATM129 envelope glycoproteins. The TEV protease cleavage site and the His10 tag are shown fused to TM residue 129. C, Western immunoblotanalysis of SUATM129 purified from two negative cell lines (lanes 1 and 2) and two cell lines expressing SUATM129 (lanes 3 and 4). Samples were either leftnonreduced or reduced with �-mercaptoethanol and separated by SDS-PAGE, or left in the native state and separated by PAGE using Criterion 4 –15% gradientgels. The proteins were transferred to nitrocellulose and the Western immunoblots probed first with either an anti-His or anti-SUA monoclonal antibody, andthen a peroxidase-conjugated goat anti-mouse IgG. The bound complexes were visualized by chemiluminescence. Molecular mass markers are in kDa.D, SDS-PAGE analysis of two different purified SUATM129 preparations (preparation 1 in lanes 1 and 2; preparation 2 in lanes 3 and 4). Nonreduced SUATM129samples after metal affinity purification (lanes 1 and 3) or after metal affinity plus octyl-Sepharose purification (lanes 2 and 4) were separated by SDS-PAGE usingCriterion 4 –15% gradient gels. The proteins were visualized with silver stain. Molecular mass markers (lane M) are in kDa.




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to analysis by nanoLC-ESI-MS/MS. The fractions that wereanalyzed by chip-based direct infusion FTICR-ECD-MS wereredried and solubilized in 0.2%N-ethylmorpholine buffer plus 1ng of Asp-N and digested at 37 °C for 1.5 h.Chip-based Infusion-FTICR-MS with ECD—TheHPLC frac-

tions were resolubilized in 30 �l of 40% acetonitrile, 0.2% for-mic acid, 5% isopropyl alcohol. All disulfide-linked peptideswere identified by screening the fractions with chip-based ESIinfusion using an Advion Nanomate 100 coupled to a Ther-moFinnigan LTQ-FT Hybrid Ion Cyclotron Resonance MassSpectrometer upgraded with the ECD capability. The Nano-mate was set to pick up 6 �l of sample and spray at 1.5 kV withN2 back-pressure of 0.3 pound/square inch. Each fraction wasscreened for 3 min, performing data-dependent acquisitionsconsisting of a FT full scan from 300 to 2000 m/z with theresolving power set at 100,000 @ 400 m/z followed by an ECDscan from 100–2000 m/z (3 microscans, 100,000 R, energy �4.0, delay � 10 ms, duration � 100 ms) on ions with chargestates of [M�2H]2� or higher, plus a linear ion trap collision-induced dissociation (CID) scan on ions with charge states of[M�2H]2� and [M�3H]3�. Each ion was analyzed twice thenplaced on an exclusion list for 60 s. All ECD spectra were ana-lyzed manually by looking for the presence of theoretical tryp-sin-cleaved peptide ions. Non-cysteine-containing peptide ionswere identified from analysis of the CID-MS/MS spectra. Ther-moFinnigan Xcalibur QualBrowser software was used to deter-mine the theoretical isotopic distributions for the isotopeclusters.Orbitrap CID Fragmentation—Fractions containing pep-

tides with adjacent cysteines were analyzed by nanoLC-ESI-MS/MS using a ThermoFinnigan LTQ Orbitrap Hybrid MassSpectrometer (Thermo Fisher Scientific) coupled to a nanoLC-two-dimensional HPLC system (Eksigent, Dublin, CA). Thechymotrypsin fraction containing the C1 and C17C18C19 pep-tides was treated with Asp-N protease, after which an aliquotwas diluted with 0.15% formic acid and 0.05% TFA and loadedonto a 250-nlOPTI-PAK trap (OptimizeTechnologies,OregonCity, OR) custom packed with Michrom Magic C8 solid phase(Michrom Bioresources, Auburn, CA). Chromatography wasperformed using 0.2% formic acid in both the A solvent (98%water and 2% acetonitrile) and B solvent (80% acetonitrile, 10%isopropyl alcohol, and 10% water), and a 10% B to 50% B gradi-

ent over 45 min at 325 nl/min through a hand-packed PicoFrit(NewObjective,Woburn,MA)nanobore (MichromMagicC183-�m 75 �m � 200 mm) column. The LTQ Orbitrap massspectrometer experiment consisted of a FT full scan from 300to 1200m/zwith resolution set at 60,000 (at 400m/z), followedby Orbitrap CID MS/MS scans on the top three ions at 30,000resolution. Dynamic exclusion was set to 2, and selected ionswere placed on an exclusion list for 30 s. The lock-mass optionwas enabled for the FT full scans using the ambient air polydi-methylcyclosiloxane ion of m/z � 445.120024 or a commonphthalate ionm/z � 391.284286 for real-time internal calibra-tion (35). The MS/MS spectra ions were assigned manually.Edman Degradation N-terminal Chemical Sequencing—Rel-

ative abundance of the disulfide-linked peptides was deter-mined for some of the HPLC fractions using Edman degrada-tion N-terminal chemical sequencing on a Procise cLC ProteinSequencer (Applied Biosystems, Foster City, CA). The aminoacid assignments were made manually, and the relativeamounts of each peptide were determined from the amino acidyields of the first few sequencing cycles.

RESULTS

SUATM129 Protein—The SUATM129 glycoprotein wasproduced in chicken cells, normal host cells for ASLV replica-tion, to ensure appropriate envelope glycoprotein folding, gly-cosylation, and transport. DF-1 cell lines were established thatstably produced SUATM129. SUATM129 was expressed as asecreted protein with a histidine tag to allow a relatively simplepurification procedure of biologically active protein from cellculture supernatants in good yield (see “Experimental Proce-dures”). Biological activity was demonstrated by the ability ofthe SUATM129 protein to bind a soluble form of the ASLVsubgroup A Tva receptor as shown by immunoprecipitation(data not shown) and the ability of SUATM129 to inhibit cellsurface expressed Tva and specifically block ASLV(A) infectionof normally susceptible cells by greater than 10–50-fold com-pared with another ASLV subgroup (data not shown). The cal-culated molecular mass of SUATM129 is 53,496 Da; the SUAsubunit (37,265 Da) connected to the TM129 subunit (16,249Da) by a disulfide bond. Previously published work on thesecreted SUA-rIgG immunoadhesin, the complete SUA sub-unit fused to a rabbit IgGdomain, identified that 10 of 11 poten-tial N-linked glycosylation sites in SUA were glycosylated with�23 kDa of carbohydrate. If the SUATM129 glycoprotein con-tains similar levels of glycosylation, the SUATM129 glycosy-lated molecular mass would be at least 76,496 Da; the SU sub-unit �60,264 Da. The ASLV TM subunit has two possible sitesof N-linked glycosylation that could add additional carbohy-drate to the SUATM129 glycoprotein.The SUATM129 expressed by cell lines was initially charac-

terized by small scale purification using a nickel affinity resin tobind the His tag and the eluted proteins analyzed by PAGE andWestern immunoblots using an antibody against the HIS-tagand an antibody that specifically binds the SUA subunit. Pro-teins were either reduced or left nonreduced before SDS-PAGEto break or maintain the disulfide bond linking SUA and TM,respectively, or the proteins were left in the native state andseparated by PAGE. Fig. 1C is an example of this analysis. The

TABLE 1HPLC running conditions for peptide separation

Protease Gradient Time % B

minSUATM/trypsin Phase A: 5% acetonitrile/0.1% TFA

Phase B: 80% acetonitrile/0.1% TFA 0 515 5115 45117 80120 80122 5135 5

SUATM/chymotripsin Phase A: 5% acetonitrile/0.1% TFAPhase B: 80% acetonitrile/0.1% TFA 0 2

5 210 859 3069 8072 8073 278 2




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nonreduced SUATM129 protein migrated at �85 kDa, withthe reduced protein yielding the SUA subunit at �75–80 kDaand the TM129 at �20 kDa. The native gel showed that at leastsome of the SUATM129 glycoprotein was migrating as an oli-gomeric form or forms as predicted from the initial report onthis protein. Large scale purification of SUATM129 yielded aprotein mixture predominantly consisting of SUATM129 butalso containing several other proteins (Fig. 1D, lanes 1 and 3).Analysis of the protein bands from the SDS-PAGE separatednickel-resin-purified protein preparation by mass spectrome-try identified fibronectin, lactate dehydrogenase, and lamin Aas the major contaminants (data not shown). This initial puri-fied product was further purified using octyl-Sepharose chro-matography that improved the overall purity of the finalSUATM129 product (Fig. 1D, lanes 2 and 4). The SUATM129glycoprotein preparation purified by metal affinity chromatog-raphy followed by octyl-Sepharose chromatography was usedfor all subsequent experiments.HPLC Analysis of SUATM129 Peptides—The SUATM129

glycoprotein presented several challenges for enzymatic diges-tion andHPLCof the peptides due to carbohydrate content andthe desire to maintain the disulfide bonds. Attempts to removethe carbohydrate from intact SUATM129 prior to enzymaticdigestion resulted in the precipitation of the deglycosylatedprotein that was difficult to resolubilize. Subsequent enzymatic

digests resulted in chromatogramswith inconsistent, poor peakshapes (data not shown). Better chromatography performancewas observedwhen SUATM129was first digested into peptideswith a protease and then deglycosylated with PNGase F. Theupper panel in Fig. 2 shows the HPLC chromatogram ofSUATM129 following trypsin digestion and deglycosylation,with the disulfide bonds intact. Half of the same digest mixturewas then reduced with TCEP and run under identical HPLCconditions (lower panel). The chromatogram for the nonre-duced mixture clearly shows peaks that are missing from thechromatogram of the disulfide-reduced digestmixture. It is notclear in the TCEP-treated chromatogram where the reducedcysteine peptides are eluting. The fractions corresponding tothe nonreduced peaks contain disulfide-linked peptides thatwere initially used to develop the automated chip-based directinfusion electrospray FTICRmethod on the LTQ-FT. Once themethodswere established, allHPLC fractions from the proteasedigests were screened and analyzed.FTMS Analysis of SUATM129 Peptide Pairs—Previous stud-

ies have shown that performing ECD on disulfide-paired pep-tides cleaves the disulfide bond more readily than the peptidebackbone amide bonds (36, 37). The LTQ-FT with the ECDoption is a hybrid mass spectrometer that uses the linear iontrap for the controlled isolation of an ion and the FTICR cell toperform ECD on that ion, scanning the resulting fragments

FIGURE 2. Reverse-phase HPLC chromatograms of nonreduced or reduced, trypsin-digested and deglycoslyated SUATM129 peptides. Some of thepeptide peak changes between the nonreduced and reduced chromatograms, presumably due to the breaking of a disulfide bonded peptide pair, arehighlighted with arrows.




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with highmass accuracy. In this study, we used chip-based infu-sionwith theAdvionNanomate on an LTQ-FT to develop ECDconditions that minimized peptide bond fragmentation andgive ECD fragment spectra containing both the intact disulfide-paired ion and the disulfide-cleaved peptide ions. These condi-tions were used in the LTQ-FT data-dependent method toscreen the HPLC fractions from the nonreduced SUATM129protease digests. The method provided accurate full scan massdata and accurate mass ECD spectra giving predominantly theparent ion corresponding to the mass of the disulfide linkedpeptide pair and the ions of the dissociated peptides as frag-ment ions. The ions weremanually compared with the theoret-ical ions corresponding to protease-specific SUATM129 cys-teine-containing peptides. The FT full scan and the ECDspectrum in Fig. 3 illustrate the method used to assign the C3and C4 peptides as a disulfide-bonded pair. The C3�C4 ion835.41901 [M�3H]3� was isolated (Fig. 3A) and cleaved withECD, and the resulting ions were analyzed identifying the orig-inal C3�C4 ion, the C3 ion 1602.68994 [M�H]1�, and the C4ion 902.56372 [M]�� (Fig. 3B). Themechanismof ECDdisulfidebond cleavage often results in one of the peptides acquiring ahydrogen atom on the cysteine sulfur while the other peptidecysteine sulfur does not (36, 37). Analysis of the ECD-cleavedC3 and C4 ions shows an isotopic distribution of the singlycharged C4 peptide ion with a cysteine sulfur that did notacquire a hydrogen, whereas the C3 peptide ion was proto-nated, though not completely (Fig. 3C).A summary of the FTICR-ECD analyses of the HPLC frac-

tions containing the nonreduced digest peaks is shown in Fig. 4.The differences between the observed (measured) peptidemasses, both parent ion and ECD-generated ions and the the-oretical (calculated) masses, are reported as delta mass in partsper million (35). Four fractions from the nonreduced trypsindigest were found to contain a single peptide pair with the ECDspectra demonstrating that C3 is bound to C4 in fraction 57, C7is bound toC8 in fraction 50, C13 is bound toC14 in fraction 87,and C15 is bound to C16 in fraction 67 (data not shown).The FT-MS full scan of fraction 112 contained a single pre-

dominant ion 1258.93970 [M�6H]6�. The ECD spectra on thision did not produce any useful ions, in part, due to the overalllow intensity of the parent ion and the large size of the C5peptide. We hypothesized that this ion may be the C5 and C6peptide disulfide pair based on the expected mass. Attemptsusing a second protease to decrease the length of the presumedC5 peptide failed to produce any clear information, so the frac-tion was treated with TCEP to see whether the 1259 ionresponded to reduction. Another FT-MS full scan of the TCEP-treated fraction 112 yielded multiply charged ions correspond-ing to C5 and C6, demonstrating that C5 is bound to C6 inSUATM129. The observed peptide masses and isotopic massdistributions were all in close agreement with their theoretical

masses (Fig. 4) and theoretical isotopic distributions (data notshown).Fraction 74 contained the ion 1010.05450 [M�5H]5�, which

produced an ECD spectrum with a 726.88186 [M�2H]2�� ioncorresponding to the C9 peptide and a 1797.74756 [M�2H]2 � 3�

ion corresponding to the C10, C11, and C12 peptide (data notshown). It is unclear from these data as towhich cysteine theC9peptide was linked although it was unlikely to be C10 becausethat would mean the adjacent cysteines C11 and C12 would bea disulfide-bonded pair. We noted that there was an asparticacid between C9 and C10 and one between C10 and C11 so theremainder of that fraction was treated with Asp-N proteasegenerating three disulfide-linked peptide chains: one peptidewith C9, one with C10, and one peptide with C11C12. Subse-quent MS analysis detected the ion 736.07969 [M�4H]4� cor-responding to all three peptides bonded together, ruling out C9being disulfide-linked to C10 (data not shown). The ECD cleav-age spectra of the [M�4H]4� ion at m/z 736.07969 containedthe expected masses corresponding to the C9 peptide, the C10peptide, and the C11C12 peptide (Fig. 5). The observed peptidemasses and isotopicmass distributions were in close agreementwith the theoretical masses (Fig. 4) and theoretical isotopic dis-tributions (data not shown). These data allowed us to concludethat C9 is bound to either C11 or C12 with C10 bound to theopposite cysteine.Analysis of the trypsin digest HPLC fractions did not yield

any usefulMSdata for the assignments of theC1, C2 peptide, ortheC17,C18,C19peptide.We identified theC1,C2�C17,C18,C19 peptide pair only by Edman chemical sequencing (Fig. 4).Therefore, a chymotrypsin digest followed by PNGaseF treat-ment was performed on a new portion of the purifiedSUATM129 protein. The digest was fractionated by reverse-phase HPLC, and fractions were analyzed by ECD-MS similarto the trypsin digest. The FT full scan of chymotrypsin HPLCfraction 41 contains a 694.92413 [M�3H]3� ion that corre-sponds to the theoretical mass of the C1 and C17, C18, C19peptide pair connected with a disulfide bond (Fig. 6A). Theobserved mass and isotopic mass distribution were in closeagreement with the theoretical mass (� mass �0.8 parts permillion) and theoretical isotopic distribution. The spectra fromECD cleavage of the [M�3H]3� ion contained the parent[M�3H]3� ion plus two singly charged ions correspondingto the C1 peptide ([M�H]1� at m/z 551.24976) and theC17C18C19 peptide ([M�H]1 � 3� atm/z 1533.53149) (Fig. 6B).The observed peptidemasses and isotopicmass distributions ofthese peptides were in close agreement with their theoreticalmasses (Fig. 4) and theoretical isotopic distributions (data notshown). Similar to the C9, C10, C11, and C12 assignments dis-cussed above, it was not clear where the C1 peptide disulfidewas linked to the C17, C18, C19 peptide. Again, there is anaspartic acid between C17 and C18, and C18, C19 are adjacent

FIGURE 3. Example of the mass spectrometry analysis of a disulfide-bonded peptide pair using ECD. A, FTICR full scan of the HPLC separation oftrypsin-digested SUATM129 protein fraction 57 is shown. The ion with an exact mass corresponding to the C3 peptide and the C4 peptide connected by adisulfide bond is indicated. The isotopic distribution of the peptides is shown in the inset. B, ECD scan triggered from the 835.41901 [M�3H]3� ion is shown.Experimental conditions were used so that each of the singly charged ECD fragment ions as well as the parent ion were present in the ECD spectra. The singlycharged ECD fragment ions add up to the mass of the parent ion. The isotopic distributions of the ECD fragment peptides are shown in the insets. C, ECDfragmentation can produce peptides with variable protonation. The isotopic distribution of the ECD fragment peptides shows that the resulting cysteinesulfurs can be partially protonated (C3) or completely deprotonated (C4) compared with the theoretical distributions.




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cysteines. A secondary digestion of a portion of fraction 41withAsp-N protease, followed by FT-MS analysis showed a706.26166 [M�3H]3� ionwhich corresponds to the theoreticalmass of the three disulfide-linked peptides (Fig. 4; data notshown). These data indicate that C1 is not linked to C17 andmust therefore be linked to C18 or C19, which suggests SUA islinked to TM through C1 and either C18 or C19, and C17 isbound to the opposite cysteine.CIDOrbitrapMSAnalysis—SUATM129 contains two sets of

adjacent cysteine pairs, C11 and C12 in SUA, and C18 and C19in TM (Fig. 1A). In an effort to try and assign the specific disul-

fide bonds to these adjacent cysteine pairs, Orbitrap CIDMS/MS analysis was employed in the hope of creating accuratemass fragments generated from cleavage of the peptide bondbetween the adjacent cysteines that still had an intact disulfidebond. Working with a chymotrypsin fraction containing C1and C17, C18, C19 peptides, we used the Orbitrap for the FTfull scan and the analysis of the MS/MS fragments (Fig. 7A). Inthis experiment the methionine in the peptide pair was oxi-dized due to storage. The FT spectrum contains the 2�, 3�,and 4� charge states of the disulfide-linked C1 peptide and theC17, C18, C19 peptide. The three possible disulfide pairings are

FIGURE 4. Summary of the MS analysis of disulfide-bonded peptide pairs of SUATM129. The accurate mass measurements of the disulfide-bondedpeptide pairs were made using FTICR mass spectrometry with an LTQ-FT. The measured masses are shown as mass/ion charge (m/z). ECD was used topreferentially break the disulfide bond of the peptide pair to yield the original peptide pair, and each peptide mass was measured (e.g. ECD C3; ECD C4). Onedifference to note between using ECD compared with chemical reduction of a disulfide bond: ECD does not necessarily result in peptides with replacedhydrogens on all of the now free sulfur atoms. Peptides generated by ECD that have (�) or have not (�) replaced the hydrogen ions are indicated in the H�column. For peptide pair C5�C6, the fraction was reduced and analyzed by LTQ-FTICR MS to measure the masses of the reduced peptides (redC5; redC6).Peptide pairs from CID that define the disulfide bond pattern between cysteines C1 and C17,18,19 are shown with the complete data presented in Fig. 7. TheC1, C2�C17, C18, C19 peptide pair was identified only by Edman chemical sequencing. The analytical difference between the observed mass compared withthe theoretical mass is given as Delta Mass in parts per million. The peptide sequences corresponding to the measured mass are shown with the putativedisulfide bonds. The SUATM129 glycoprotein contains two adjacent cysteine pairs: C11,C12 and C18,C19 (marked in boxes). The methionine in the C17, C18,C19 peptide in Fraction 41 was found to be oxidized (oxM) after freeze/thaw from storage: this was the predominant form in the Asp-N and CID analysis. Weconsistently observed an unusual chymotrypsin cleavage between Gln29 and Ser30 generating the C1 peptide CLSTQ in multiple digests. We have no definitiveexplanation, but it may reflect the extended overnight digestion at room temperature.




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also shown. The Orbitrap performed CID MS/MS on the1049.88477 [M�2H]2� ion; the MS/MS spectrum is shown inFig. 7B. We generated a list of theoretical b and y ion seriesmasses with the disulfide bonds intact and fragmentation ofeach peptide if the cleavage occurred betweenC18 andC19 andlooked for those masses manually in the accurate massMS/MSspectra (data not shown). There weremany ions correspondingto fragments with C18 and C19 intact, but there was also asingly charged ion at 1282.44421 that matched a peptide frag-ment with C17 linked to C18. There was also a singly chargedion at 817.32465 that corresponds to peptides with C1 linked toC19 (Figs. 4 and 7B). Both of these fragments are within 4 partspermillion of the theoretical fragment pairmasses and are gen-erated from a parent ion that corresponds to the C1 and C17,C18, C19 chymotrypsin peptide pair, so we propose that C1 isdisulfide-linked to C19 and C17 is linked to C18. We alsoattempted to use this approach to assign the disulfide pairingfor the C9 and the C10, C11, C12 linked peptides, but wereunsuccessful: no peptides were identified resulting fromcleaved between the adjacent C11 and C12 residues.N-Linked Glycosylation Pattern of SUATM129—The

SUATM129 protein contains 13 possible N-linked glycosyla-tion sites (NXS/T): 11 sites in SU and 2 sites in TM (Fig. 1A).Because PNGase F treatment of an asparagine modified withcarbohydrate converts the residue to an aspartic acid, the vari-ous high resolution mass spectra provided the data to assesswhich of these possible sites in the SUATM129 protein were infact modified with carbohydrate. The masses and sequencescorresponding to the peptides that contain a cysteine residueand a possible N-linked glycosylation site with either anunmodified asparagine or an asparagine modified with carbo-hydrate converted to an aspartic acid are shown in Fig. 4. Pep-tides containing the other possible glycosylation sites were

identified in the mass spectra (data not shown). All of the MSanalysis performed consistently found all possibleN-linked gly-cosylation sites of SUATM129 to be modified with carbohy-drate except sites N8 andN13 (Fig. 8). In a previous study of therelated SUA-rIgG protein, a secreted protein with the SUA gly-coprotein fused to a rabbit IgG domain, 10 of 11 possibleN-linked glycosylations sites were modified with carbohydrate:as with the SUATM129 protein, the exception was N8 (38).

DISCUSSION

We describe a novel method that enabled the assignment ofmultiple disulfide-linked peptides from a single HPLC separa-tion of a nonreduced digest of a complex glycoprotein(SUATM129) using accurate mass measurements of the disul-fide-paired ions and ECD-generated peptides with commer-cially available instrumentation (LTQFT and Nanomate). Thedata acquisition was easy to automate and allowed for hands offscreening of a large number of HPLC fractions. Experimentalconditions were developed to maximize the cleavage of thedisulfide bonds while minimizing peptide backbone cleavage,which gives very distinctive ECD spectra from the disulfide-linked peptides. Because the ECD fragment ionmasses will addup to the parent ion mass taking into account the addition of ahydrogen, this made it a simple task to scan through and findcysteine-paired peptides manually, especially for single pairedpeptides. Certainly a software-assisted approach similar tomatching peptide identification could be employed to auto-mate the process totally. Using just this approach, we were ableto assign four disulfide pairs from the complex SUATM129protein using a single HPLC run. Combined with secondaryprotease digestions and additional accuratemass spectrometry,we were able to assign nearly all (7 of 9) disulfide bonds as well

FIGURE 5. LTQ-FTICR ECD MS analysis of the 4� ion of the SUATM129 trypsin�Asp-N C9�C10�C11, C12 disulfide-bonded peptides, mass 736.07969.MS analysis of the ECD reaction shows the 4� ion mass of the original bonded peptides and each individual peptide: the peptide (z � 1) containing C9, thepeptide (z � 1) containing C10, and the peptide (z � 1) containing C11 and C12.




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as identify the carbohydrate-modified N-linked glycosylationsites of SUATM129 using these methods (Fig. 8).It should be noted that in screening the HPLC fractions, a

number of alternate cysteine pairs could be detected from lowerbackground level signals. Recognizing the possibility of disul-

fide rearrangement during protein purification and the prote-ase digestion steps despite great effort to keep these bonds sta-ble, we relied on the UV absorbance of the HPLC peaks todetermine that we were analyzing the most abundant cysteine-paired components in the digests. In addition, we repeatedly

FIGURE 6. LTQ-FTICR MS analysis of the SUATM129 chymotrypsin peptide pair containing cysteine C1 and cysteines C17,18,19. A, full scan of thechymotrypsin digest of SUATM129 HPLC fraction 41 is shown with accurate mass measurement of the 3� ion (z � 3) of the C1 and C17, C18, C19 peptide pair,mass � 694.92413. The actual isotopic distribution and the theoretical isotopic distribution of mass 694.92413 are shown as insets. B, LTQ-FTICR ECD MSanalysis of mass 694.92413 corresponding to the 3� ion of the SUATM129 chymotrypsin C1 and C17, C18, C19 disulfide-bonded peptide pair and eachindividual peptide 1� ion from the parent. The isotopic distributions of the ECD C1 and C17, C18, C19 peptides are shown in insets.




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detected the same dominant disulfide pairs in repeat experi-ments using two different proteases to digest the protein (datanot shown). Because MS analysis does not necessarily corre-spond to peptide abundance, several cycles of Edman chemicalsequencing were performed on peptides in the HPLC fractionsto confirm that the expected disulfide-linked peptides were thedominant component of that fraction (data not shown). Finally,similar MS studies on the SUA fragment of SUA-rIgG (38) alsoidentified the same disulfide-bonded peptide pairs found in theSUA glycoprotein region of this study (data not shown).Using SUATM129 as a representative, biologically active, but

secreted protein, we propose a model of the disulfide bond andthe N-linked glycosylation patterns of the subgroup A ASLVenvelope glycoprotein (Fig. 8). As predicted from publisheddata, the SUA glycoprotein subunit is bound to the TM glyco-protein subunit by a covalent disulfide bond: we found the firstcysteine residue in SUA, C1 (Cys25), bound to the last extracel-lular cysteine in TM, C19 (Cys438). Six intrasubunit disulfidebonds define the secondary structure of the SUA glycoprotein,organizing the hr1, hr2, and vr3 variable domains for the criticalinteractions with their respective receptor to initiate entry. Adisulfide bondwas definitively assigned betweenCys60 (C3) andCys91 (C4), Cys131 (C5) and C6 (Cys184), C7 (Cys197) and C8(Cys206), and C13 (Cys292) and C14 (Cys309). Disulfide bondscould not be definitively assigned to the adjacent cysteines inSUA, C11 (Cys258) and C12 (Cys259). The data show that a pep-tide containing C9 (Cys221) was bound to a peptide that con-tained C10 (Cys247), C11 and C12 and that further proteindigestion proved that C9was not bound to C10. Thus, there is adisulfide bond betweenC9 andC11 orC12, and a bond betweenC10 and the remaining C11/C12 residue. Two intrasubunitdisulfide bonds were assigned in the TM glycoprotein: betweenC15 (Cys348) and C16 (Cys385), and C17 (Cys430) and C18

(Cys437). This proposed disulfide bond pattern leaves C2(Cys38) as a free cysteine.In this study, we have characterized a secreted form of the

subgroupAASLVenvelope glycoprotein. Because the positionsof the cysteine residues and theN-linked glycosylation sites areremarkably conserved between the subgroup A to E ASLV gly-coproteins, we believe that the secondary structures of theseglycoproteins will also be conserved including the position ofthe free cysteine C2, Cys38. The exact function of this free cys-teine in ASLV fusion and entry is currently unknown. Smithand Cunningham found that binding of the subgroup A ASLVenvelope glycoprotein with the Tva receptor induced the for-mation of a cysteine thiolate (Cys-S-) at the Cys38 position (28).They found that chemical inactivation of the Cys-S- or geneticsubstitution of Cys38 completely blocked ASLV fusion andinfection. However, they determined that the Cys-S- does notmediate isomerization of the SUA and TM disulfide bond, noris it required for the interaction of TMwith a target membraneor the formation of helical bundles at low pH. Thus, some otherstep of the fusion process is dependent on the receptor-depen-dent formation of the cysteine thiolate.In an earlier study, we mapped the modified N-linked glyco-

sylation sites of the SUA glycoprotein fused to a rabbit IgGdomain (SUA-rIgG) produced in DF-1 cells and purified fromthe cell culture supernatant using mass spectrometry (38). Tenof the 11 N-linked glycosylation sites were modified in SUA-rIgG, with only N8 (Asp236) unmodified. The SUATM129 pro-tein contains both the SUA glycoprotein, and the extracellularregion of the TM glycoprotein, had 11 of the 13 possibleN-linked glycosylation sites modified by carbohydrate asdetected by the conversion of a modified asparagine to asparticacid after PNGase F digestion. The same 10 sites identified asmodified in the extracellular SU glycoprotein in this study werealso modified in a similar biologically active protein SUA-rIgG:only N8 (Asp236) was not modified with carbohydrate. One ofthe two possible N-linked glycosylation sites in the TM glyco-protein was modified: N12 (Asp392). Asp440 (N13) was notmodified with carbohydrate possibly due to its close proximityto the CX6CC motif in the linker between the heptad repeats.Glycosylation of this site may inhibit the proposed flexibility ofthis region thought necessary for the conformational changesoccurring during fusion.It has been noted that although the amino acid sequences of

the fusion glycoproteins of the Ebola virus GP2 and ASLV TMare very different, the organization of the secondary structuresrequired for efficient fusion of the viral and cellularmembranesare conserved (26). Both fusion proteins have cysteine residuesflanking an internal fusion peptide region near the N terminus,followed by a region critical for conformational changes leadingto fusion: two �-helical heptad repeat regions separated by aflexible linker region containing a CX6CC motif (39, 40). Inaddition, both the Ebola and ASLV envelope glycoproteins are

FIGURE 7. NanoLC-Orbitrap mass spectrometry analysis of the SUATM129 chymotrypsin fraction containing the C1 and C17,18,19 disulfide-linkedpeptides. In this experiment the methionine in the peptide pair was oxidized (oxM) due to storage. A, FT full scan of the multiply charged ion species of the C1and C17,18,19 peptides. All of the possible Cys-Cys bond configurations are shown. B, Orbitrap CID MS/MS spectra of the 1049.88477 [M�2H]2� parent ion ofthe C1 and C17, C18, C19 disulfide-linked peptides. Some of the accurate mass fragment ion assignments including the two disulfide-linked pairs resulting fromcleavage between C18 and C19 are indicated.

FIGURE 8. Proposed secondary structure of the SUATM129 heterodimerof SU and TM glycoproteins. The proposed disulfide bonds are indicted withred lines. Bonds to cysteines C11 and C12 (indicated with a box) could not beassigned further. The free cysteine is indicted, C2. The N-linked glycosylationsites that were converted to aspartic acid after PNGase F treatment, andtherefore contained carbohydrate additions, are marked in blue. The ungly-cosylated sites are underlined, N8 and N13.




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trimers of heterodimers, GP1 and GP2 for Ebola, SU and TMfor ASLV, with the heterodimers connected by a disulfide bond(41, 42). The disulfide bond pattern of the Ebola GP1/GP2 het-erodimer has been determined biochemically with some of thebonds verified by crystal structure (41–43). Previously, basedon the homology with Ebola GP2, the organization and disul-fide bond pattern of ASLVTMwere predicted to be the same asGP2. The data from this study have formally demonstrated thatboth Ebola GP2 and ASLV(A) TM glycoproteins have the samedisulfide bond pattern organizing the functional domains in thefusion glycoproteins: a disulfide bond linking the cysteinesflanking the internal fusion peptide, a disulfide bond betweenthe first and second cysteine residues of the CX6CC motif, andthe last cysteine in the CX6CC motif providing the covalentbond between the TM glycoprotein and the first cysteine of thesurface glycoprotein Ebola GP1 and ASLV SUA.The structural data presented here will be useful as a guide

for future experiments designed to study the entry and fusionmechanism of ASLV envelope glycoproteins as a model forenveloped virus entry. The organization of the putative recep-tor binding domains (hr1, hr2, and vr3), the proof of the orga-nization of the substructures in the TM fusion glycoprotein,and the identification of the disulfide bond linking the SU andTM heterodimers will aid in constructing new mutants to testfunction and to obtain atomic structure.

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and Mark J. FederspielGennett M. Pike, Benjamin J. Madden, Deborah C. Melder, M. Cristine CharlesworthAVIAN SARCOMA AND LEUKOSIS VIRUS ENVELOPE GLYCOPROTEIN

Disulfide Bond and Glycosylation Patterns of a Complex Protein: SUBGROUP A Simple, Automated, High Resolution Mass Spectrometry Method to Determine the

doi: 10.1074/jbc.M111.229377 originally published online March 23, 20112011, 286:17954-17967.J. Biol. Chem.

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simple,automated,highresolutionmassspectrometry ...viral glycoprotein-receptor interaction itself...

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